Adenovirus vectors containing cell status-specific response elements and methods of use thereof

ABSTRACT

The present invention provides adenoviral vectors comprising cell status-specific transcriptional regulatory elements which confer cell status-specific transcriptional regulation on an adenoviral gene. A “cell status” is generally a reversible physiological and/or environmental state. The invention further provides compositions and host cells comprising the vectors, as well as methods of using the vectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. Provisional PatentApplication No. 60/099,791, filed Sep. 10, 1998. The priorityapplication is hereby incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to cell transfection using adenoviral vectors.More specifically, it relates to cell status-specific replication ofadenovirus vectors in cells, regardless of tissue or cell type.

BACKGROUND ART

In spite of numerous advances in medical research, cancer remains thesecond leading cause of death in the United States. In theindustrialized nations, roughly one in five persons will die of cancer.Traditional modes of clinical care, such as surgical resection,radiotherapy and chemotherapy, have a significant failure rate,especially for solid tumors. Neoplasia resulting in benign tumors canusually be completely cured by removing the mass surgically. If a tumorbecomes malignant, as manifested by invasion of surrounding tissue, itbecomes much more difficult to eradicate. Once a malignant tumormetastasizes, it is much less likely to be eradicated.

Excluding basal cell carcinoma, there are over one million new cases ofcancer per year in the United States alone, and cancer accounts for overone half million deaths per year in this country. In the world as awhole, the five most common cancers are those of lung, stomach, breast,colon/rectum, and uterine cervix, and the total number of new cases peryear is over 6 million.

Lung cancer is one of the most refractory of solid tumors becauseinoperable cases are up to 60% and the 5-year survival is only 13%. Inparticular, adenocarcinomas, which comprise about one-half of the totallung cancer cases, are mostly chemo-radioresistant. Colorectal cancer isthe third most common cancer and the second leading cause of cancerdeaths. Pancreatic cancer is virtually always fatal. Thus, currenttreatment prospects for many patients with these carcinomas areunsatisfactory, and the prognosis is poor.

Hepatocellular carcinoma (HCC or malignant hepatoma) is one of the mostcommon cancers in the world, and is especially problematic in Asia.Treatment prospects for patients with hepatocellular carcinoma are dim.Even with improvements in therapy and availability of liver transplant,only a minority of patients are cured by removal of the tumor either byresection or transplantation. For the majority of patients, the currenttreatments remain unsatisfactory, and the prognosis is poor.

Breast cancer is one of the most common cancers in the United States,with an annual incidence of about 182,000 new cases and nearly 50,000deaths. In the industrial nations, approximately one in eight women canexpect to develop breast cancer. The mortality rate for breast cancerhas remained unchanged since 1930. It has increased an average of 0.2%per year, but decreased in women under 65 years of age by an average of0.3% per year. See e.g., Marchant (1994) Contemporary Management ofBreast Disease II: Breast Cancer, in: Obstetrics and Gynecology Clinicsof North America 21:555-560; and Colditz (1993) Cancer Suppl.71:1480-1489.

Despite ongoing improvement in the understanding of the disease, breastcancer has remained resistant to medical intervention. Most clinicalinitiatives are focused on early diagnosis, followed by conventionalforms of intervention, particularly surgery and chemotherapy. Suchinterventions are of limited success, particularly in patients where thetumor has undergone metastasis. There is a pressing need to improve thearsenal of therapies available to provide more precise and moreeffective treatment in a less invasive way.

Prostate cancer is the fastest growing neoplasm in men with an estimated244,000 new cases in the United States being diagnosed in 1995, of whichapproximately 44,000 deaths will result. Prostate cancer is now the mostfrequently diagnosed cancer in men. Prostate cancer is latent; many mencarry prostate cancer cells without overt signs of disease. It isassociated with a high morbidity. Cancer metastasis to bone (late stage)is common and is almost always fatal.

Current treatments include radical prostatectomy, radiation therapy,hormonal ablation and chemotherapy. Unfortunately, in approximately 80%of cases, diagnosis of prostate cancer is established when the diseasehas already metastasized to the bones, thus limiting the effectivenessof surgical treatments. Hormonal therapy frequently fails with time withthe development of hormone-resistant tumor cells. Althoughchemotherapeutic agents have been used in the treatment of prostatecancer, no single agent has demonstrated superiority over itscounterparts, and no drug combination seems particularly effective. Thegenerally drug-resistant, slow-growing nature of most prostate cancersmakes them particularly unresponsive to standard chemotherapy.

A major, indeed the overwhelming, obstacle to cancer therapy is theproblem of selectivity; that is, the ability to inhibit themultiplication of tumor cells, while leaving unaffected the function ofnormal cells. For example, in prostate cancer therapy, the therapeuticratio, or ratio of tumor cell killing to normal cell killing oftraditional tumor chemotherapy, is only 1.5:1. Thus, more effectivetreatment methods and pharmaceutical compositions for therapy andprophylaxis of neoplasia are needed.

Solid tumors frequently contain regions that are poorly vascularized,partly because the tumor cells grow faster than the endothelial cellsthat make up the blood vessels. Tumor cells can remain viable in suchhypoxic conditions and are often refractory to chemotherapy andradiation therapy. In a recent study of cervical cancer, the oxygenstatus of a tumor was shown to be the single most important prognosticfactor, ahead of age of patient, menopausal status, clinical stage, sizeand histology. Hoeckel et al. (1996) Semin. Radiat. Oncol. 6:1-8.

Of particular interest is development of more specific, targeted formsof cancer therapy, especially for cancers that are difficult to treatsuccessfully. In contrast to conventional cancer therapies, which resultin relatively non-specific and often serious toxicity, more specifictreatment modalities attempt to inhibit or kill malignant cellsselectively while leaving healthy cells intact. Radioresistant andchemoresistant tumors present particular challenges, and there is a needfor methods of treating these types of tumors.

One possible treatment approach for many of these cancers is genetherapy, whereby a gene of interest is introduced into the malignantcell. Various viral vectors, including adenoviral vectors, have beendeveloped as vehicles for gene therapy. The virtually exclusive focus indevelopment of adenoviral vectors for gene therapy is use of adenovirusmerely as a vehicle for introducing the gene of interest, not as aneffector in itself. Replication of adenovirus has been viewed as anundesirable result, largely due to the host immune response. In thetreatment of cancer by replication-defective adenoviruses, the hostimmune response limits the duration of repeat doses at two levels.First, the capsid proteins of the adenovirus delivery vehicle itself areimmunogenic. Second, viral late genes are frequently expressed intransduced cells, eliciting cellular immunity. Thus, the ability torepeatedly administer cytokines, tumor suppressor genes, ribozymes,suicide genes, or genes which convert prodrug to an active drug has beenlimited by the immunogenicity of both the gene transfer vehicle and theviral gene products of the transfer vehicle as well as the transientnature of gene expression.

Use of adenoviral vectors as therapeutic vehicles for cancer has beenreported. Some of these approaches utilize tissue (i.e., cell type)specific transcriptional regulatory elements to selectively driveadenoviral replication (and thus cytotoxcity). U.S. Pat. No. 5,698,443;see also WO 95/11984; WO 96/17053; WO 96/34969; WO 98/35028. Whileuseful and promising, there remain other treatment contexts for whichtissue specific replication may be insufficient:

Besides cancerous cells, it is often desirable to selectively destroycertain unwanted cells or tissues. Besides surgery, however, which isinvasive, there is a dearth of methods available, particularlynon-invasive methods, which would allow such selective cytotoxicityand/or suppression.

There is a need for vector constructs that are capable of eliminatingessentially all cancerous cells in a minimum number of administrationsbefore specific immunological response against the vector preventsfurther treatment and which are suitable for use in specific, focusedcancer ablation treatments. There is also a need for an ability toselectively destroy, or impair, unwanted cells, regardless of cell typeand/or regardless of anatomical location.

SUMMARY OF THE INVENTION

Replication-competent adenoviral vectors specific for cells in a given,or particular, physiological state that permits or induces expression ofpolynucleotides under transcriptional control of a cell status-specificTRE, and methods for their use are provided. In thesereplication-competent adenovirus vectors, one or more adenoviral genesis under transcriptional control of an cell status-specifictranscriptional regulatory element (TRE). Preferably, the adenoviralgene under transcriptional control of a cell status-specific TRE is onethat is essential for adenoviral propagation. A transgene under controlof the cell status-specific TRE may also be present. For the adenoviralvectors of the present invention, a cell status-specific TRE is activein a cell existing in a particular, reversible, physiological state,which may be an aberrant physiological state, i.e., a physiologicalstate that deviates from the typical, or normal, physiological state ofthat same cell when in a non-dividing or regulated dividing state undernormal, physiological conditions.

Accordingly, in one aspect, the invention provides an adenovirus vectorcomprising an adenovirus gene, wherein said adenovirus gene is undertranscriptional control of a cell status-specific TRE. In anotherembodiment, a cell status-specific TRE is human. In another embodiment,a cell status-specific TRE comprises a cell status-specific promoter andenhancer. In yet another embodiment, a cell status-specific TRE isjuxtaposed with a cell type-specific TRE, and together the two elementscontrol expression of an adenovirus gene. Thus, the invention providesadenovirus vectors comprising a TRE comprising a cell status-specificTRE and a cell type-specific TRE.

In some embodiments, the adenovirus gene under transcriptional controlof a cell status-specific TRE is an adenovirus gene essential forreplication. In some embodiments, the adenoviral gene essential forreplication is an early gene. In another embodiment, the early gene isE1A. In another embodiment, the early gene is E1B. In yet anotherembodiment, both E1A and E1B are under transcriptional control of a cellstatus-specific TRE. In other embodiments, the adenovirus gene essentialfor replication is a late gene.

In another embodiment, the cell status-specific TRE comprises a hypoxiaresponsive element. In another embodiment, the cell status-specific TREcomprises the nucleotide sequence of SEQ ID NO:1.

In another embodiment, the cell status-specific TRE comprises a cellcycle-specific TRE. The cell cycle-specific TRE can be derived from theE2F1 5′ flanking region. In one embodiment, the cell cycle-specific TREcomprises the nucleotide sequence depicted in SEQ ID NO:2.

In other embodiments, the adenovirus vector can further comprise atransgene, wherein said transgene is under transcriptional control of ancell status-specific TRE. In some embodiments, the transgene is acytotoxic gene.

In other embodiments, the adenoviral vector comprises an adenoviral geneessential for adenoviral replication under control of a first cellstatus-specific TRE, and a second adenoviral gene essential foradenoviral replication under control of a second cell status-specificTRE. The first and the second cell status-specific TREs can beidentical, substantially identical, or different from, one another.

In other embodiments, the adenoviral vector comprises an adenoviral geneessential for adenoviral replication under control of a first cellstatus-specific TRE, and a transgene under control of a second cellstatus-specific TRE. The first and the second cell status-specific TREscan be substantially identical to, or different from, one another.

In other embodiments, the adenovirus vector comprises an adenovirus geneunder transcriptional control of a cell status-specific TRE, and asecond adenovirus gene under transcriptional control of a celltype-specific TRE. In other embodiments, the adenovirus vector comprisesan adenovirus gene under transcriptional control of a cellstatus-specific TRE, and a transgene under transcriptional control of acell type-specific TRE.

In another aspect, the invention provides a host cell comprising theadenovirus vector(s) described herein.

In another aspect, the invention provides pharmaceutical compositionscomprising an adenovirus vector(s) described herein.

In another aspect, the invention provides kits which contain anadenoviral vector(s) described herein.

In another aspect, methods are provided for conferring selectivecytoxicity in target cells (i.e., cells which permit or induce a cellstatus-specific TRE to function), comprising contacting the cells withan adenovirus vector(s) described herein, whereby the vector enters thecell.

Another embodiment of the invention is an adenovirus which replicatespreferentially in mammalian cells whose cell status permits or inducesthe function of a cell status-specific TRE.

In another aspect, methods are provided for propagating an adenovirusspecific for mammalian cells whose cell status permits the function of acell status-specific TRE, said method comprising combining an adenovirusvector(s) described herein with mammalian cells whose cell statuspermits the function of a cell status-specific TRE, whereby saidadenovirus is propagated.

The invention further provides methods of suppressing tumor cell growth,more particularly a target tumor cell (i.e., a tumor cell that permitsor induces a cell status-specific TRE to function), comprisingcontacting a tumor cell with an adenoviral vector of the invention suchthat the adenoviral vector enters the tumor cell and exhibits selectivecytotoxicity for the tumor cell.

In another aspect, methods are provided for detecting cells whose cellstatus permits the function of a cell status-specific TRE in abiological sample, comprising contacting cells of a biological samplewith an adenovirus vector(s) described herein, and detecting replicationof the adenovirus vector, if any.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of adenovirus vector CN796, inwhich the E1A gene is under transcriptional control of an HRE and aPSA-TRE, as described in Example 1.

FIG. 2 shows the nucleotide sequence of the 5′ flanking region of ahuman E2F1 gene (SEQ ID NO: 1). The asterisk indicates the transcriptionstart site.

FIG. 3A-D depicts a nucleotide sequence of a prostate-specific antigenTRE.

FIG. 4A-I depicts a nucleotide sequence of a carcinoembryonic antigenTRE.

FIG. 5A-H depicts a nucleotide sequence of a human glandular kallikreinTRE.

FIG. 6 depicts a nucleotide sequence of a mucin TRE.

FIG. 7 depicts a nucleotide sequence of a rat probasin TRE.

FIG. 8 depicts a nucleotide sequence and translated amino acid sequenceof an adenovirus death protein.

MODES FOR CARRYING OUT THE INVENTION

We have discovered and constructed replication-competent adenovirusvectors which contain an adenoviral gene under transcriptional controlof a cell status-specific transcriptional response element (TRE) suchthat the adenovirus gene is transcribed preferentially in cells whosecell status permit the function of the cell status-specific TRE, andhave developed methods using these adenovirus vectors. In some preferredembodiments, the adenovirus vectors of this invention comprise at leastone adenovirus gene necessary for adenoviral replication, preferably atleast one early gene, under the transcriptional control of a TREspecifically regulated by binding of transcriptional factor(s) and/orco-factor(s) necessary for transcription regulated by the cellstatus-specific TRE. By providing for cell status-specific transcriptionof at least one adenovirus gene required for replication, the inventionprovides adenovirus vectors that can be used for specific cytotoxiceffects due to selective replication and/or selective transcription.This is especially useful in the cancer context, in which targeted cellkilling is desirable. This is also useful for targeted cytotoxic effectsin other, non-tumor cells, when selective destruction and/or suppressionof these cells is desirable. The vectors can also be useful fordetecting the presence of cells whose cell status permits function of acell status-specific TRE in, for example, an appropriate biological(such as clinical) sample. Further, the adenovirus vector(s) canoptionally selectively produce one or more proteins of interest in atarget cell by using a cell status-specific TRE.

We have found that adenovirus vectors of the invention replicate and/orexpress an adenoviral gene operably linked to a cell status-specific TREpreferentially in cells whose status permits the function of a cellstatus-specific TRE. In contrast to previously-described adenoviralvectors designed to replicate preferentially in specific, differentiatedcell types, the adenovirus vectors of the present invention compriseregulatory elements that are not cell type-specific. Rather, they confercell status-specific adenoviral replication and/or cell status-specificexpression of an operably linked adenoviral gene and/or transgene.

The adenovirus vectors of the present invention comprise a cellstatus-specific TRE which is functional in a cell which exhibits aparticular physiological (i.e., environmental or metabolic)characteristic which is reversible and/or progressive. The target cellmay exhibit an aberrant physiological state, such as low oxygen tension,or may be subjected to an aberrant environmental condition, such as heator ionizing radiation, in order for the cell-status TRE to function. Thereplication preference of these vectors is indicated by comparing thelevel of replication (i.e., titer) in cells in a requisite physiologicalstate or condition (for example, an aberrant physiological state) to thelevel of replication in cells not exhibiting the requisite physiologicalstate (for example, under normal physiological conditions). Thus, theinvention also uses and takes advantage of what has been considered anundesirable aspect of adenoviral vectors, namely, their replication andpossibly concomitant immunogenicity. The probability of runawayinfection is significantly reduced due to the cell status-specificrequirements for viral replication. Without wishing to be bound by anyparticular theory, the inventors note that production of adenovirusproteins can serve to activate and/or stimulate the immune system,generally and/or specifically toward target cells producing adenoviralproteins, which can be an important consideration in the cancer context,where patients are often moderately to severely immunocompromised.

The adenovirus vectors of the present invention find particular utilityin specific treatment regimens, in which the treatment is highly focusedtoward, for example, a particular cancer which might otherwise beinoperable or untreatable. An important feature of the invention is thatthe vectors are useful in these treatments regardless of the tissue orcell type of the cancer, and yet their cytotoxicity can be targeted tocertain locations.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sanbrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Wei & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994); “Current Protocols inImmunology” (J. E. Coligan et al., eds., 1991).

For techniques related to adenovirus, see, inter alia, Felgner andRingold (1989) Nature 337:387-388; Berkner and Sharp (1983) Nucl. AcidsRes. 11:6003-6020; Graham (1984) EMBO J. 3:2917-2922; Bett et al. (1993)J. Virology 67:5911-5921; Bett et al. (1994) Proc. Natl. Acad. Sci. USA91:8802-8806.

DEFINITIONS

As used herein, a “transcription response element” or “transcriptionalregulatory element”, or “TRE” is a polynucleotide sequence, preferably aDNA sequence, which increases transcription of an operably linkedpolynucleotide sequence in a host cell that allows that TRE to function.A TRE can comprise an enhancer and/or a promoter.

As used herein, the term “cell status-specific TRE” is one that conferstranscriptional activation on an operably linked polynucleotide in acell which allows a cell status-specific TRE to function, i.e., a cellwhich exhibits a particular physiological condition, including, but notlimited to, an aberrant physiological state. “Cell status” thus refersto a given, or particular, physiological state (or condition) of a cell,which is reversible and/or progressive. The physiological state may begenerated internally or externally; for example, it may be a metabolicstate (such as low oxygen), or it may be generated due to heat orionizing radiation. “Cell status” is distinct from a “cell type”, whichrelates to a differentiation state of a cell, which under normalconditions is irreversible. Generally (but not necessarily), asdiscussed herein, a cell status is embodied in an aberrant physiologicalstate, examples of which are given below.

A “normal cell status” or “normal physiological state” is the status ofa cell which exists in normal physiological conditions and which isnon-dividing or divides in a regulated manner, i.e., a cell in a normalphysiological state.

The terms “aberrant cell status” and “aberrant physiological state”,used interchangeably herein, intend a condition of a cell which is aresponse to, a result of, or is influenced by, an aberrant physiologicalcondition. An aberrant cell status is neither cell type-specific nortissue type-specific. An aberrant cell status is defined in relation toa cell of the same type which is in a non-dividing/regulated dividingstate and under normal physiological conditions.

“Normal physiological conditions” are known to those skilled in the art.These conditions may vary, depending on a cell's location in the body.For example, oxygen tension can vary from tissue to tissue. For in vitroanalyses for the purposes of determining whether a TRE is responsive todeviations from normal physiological conditions, these conditionsgenerally include an oxygen concentration of about 20% O.sub.2, and atemperature of about 37.degree. C. “Regulated cell division” is a termwell understood in the art and refers to the normal mitotic activity ofa cell. Those skilled in the art understand that normal mitotic activityvaries from cell type to cell type. For example, many terminallydifferentiated cells in tissues exhibit little or no mitotic activity,while hematopoietic cells are generally mitotically active.

An “aberrant physiological condition” or “aberrant physiological state”,as used herein, intends a condition which deviates from normalphysiological conditions, and includes, but is not limited to, aphysiological condition that is characterized by alterations in oxygenconcentration, such as hypoxic conditions; temperatures which deviatefrom physiological temperatures; a condition that triggers apoptosis;radiation, including ionizing radiation and UV irradiation; de-regulatedcell division, resulting for example, from a lack of, or insufficientamounts of, or inactivity of, a factor which controls cell division,such as, for example, retinoblastoma protein (Rb); variations in timingof cell cycle; infection with a pathogen; exposure to a chemicalsubstance; or a combination of the above-listed conditions. Anotherexample is a mutation that could, or does, exist in any cell type, i.e.,its existence does not depend on, or is not related to, thedifferentiation state of the cell.

A “target cell”, as used herein, is one that permits or induces thefunction of a cell status-specific TRE such that it effectstranscriptional activation of an operably linked polynucleotide. Atarget cell is one which exhibits a requisite physiological (orenvironmental) state, which may be an aberrant physiological state.Preferably, a target cell is a mammalian cell, preferably a human cell.A target cell may or may not be neoplastic. By transcriptionalactivation, it is intended that transcription is increased in the targetcell above the levels in a control cell (e.g., a that cell when notexhibiting a requisite physiological state (generally a normalphysiological state) by at least about 2 fold, preferably at least about5 fold, preferably at least about 10 fold, more preferably at leastabout 20 fold, more preferably at least about 50 fold, more preferablyat least about 100 fold, more preferably at least about 200 fold, evenmore preferably at least about 400 fold to about 500 fold, even morepreferably at least about 1000 fold. The normal levels are generally thelevel of activity (if any) in a cell as tested under conditions thatactivate the cell status-specific TRE, or the level of activity (if any)of a reporter construct lacking a cell status-specific TRE as measuredin a cell exhibiting the requisite physiological condition.

A “functionally-preserved” variant of a cell status-specific TRE is acell status-specific TRE which differs from another cell status-specificTRE, but still retains cell status cell-specific transcription activity.The difference in an cell status-specific TRE can be due to differencesin linear sequence, arising from, for example, single base mutation(s),addition(s), deletion(s), and/or modification(s) of the bases. Thedifference can also arise from changes in the sugar(s), and/orlinkage(s) between the bases of a cell status-specific TRE.

An “adenovirus vector” or “adenoviral vector” (used interchangeably)comprises a polynucleotide construct of the invention. A polynucleotideconstruct of this invention may be in any of several forms, including,but hot limited to, DNA, DNA encapsulated in an adenovirus coat, DNApackaged in another viral or viral-like form (such as herpes simplex,and AAV), DNA encapsulated in liposomes, DNA complexed with polylysine,complexed with synthetic polycationic molecules, conjugated withtransferrin, and complexed with compounds such as PEG to immunologically“mask” the molecule and/or increase half-life, and conjugated to anonviral protein. Preferably, the polynucleotide is DNA. As used herein,“DNA” includes not only bases A, T, C, and G, but also includes any oftheir analogs or modified forms of these bases, such as methylatednucleotides, internucleotide modifications such as uncharged linkagesand thioates, use of sugar analogs, and modified and/or alternativebackbone structures, such as polyamides. For purposes of this invention,adenovirus vectors are replication-competent in a target cell.

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include a single-,double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid,or a polymer comprising purine and pyrimidine bases, or other natural,chemically, biochemically modified, non-natural or derivatizednucleotide bases. The backbone of the polynucleotide can comprise sugarsand phosphate groups (as may typically be found in RNA or DNA), ormodified or substituted sugar or phosphate groups. Alternatively, thebackbone of the polynucleotide can comprise a polymer of syntheticsubunits such as phosphoramidates and thus can be a oligodeoxynucleosidephosphoramidate (P—NH2) or a mixed phosphoramidate-phosphodiesteroligomer. Peyrottes et al. (1996) Nucleic Acids Res. 24:1841-8;Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-23; Schultz et al.(1996) Nucleic Acids Res. 24:2966-73. A phosphorothiate linkage can beused in place of a phosphodiester linkage. Braun et al. (1988) J.Immunol. 141:2084-9; Latimer et al. (1995) Mol. Immunol. 32:1057-1064.In addition, a double-stranded polynucleotide can be obtained from thesingle stranded polynucleotide product of chemical synthesis either bysynthesizing the complementary strand and annealing the strands underappropriate conditions, or by synthesizing the complementary strand denovo using a DNA polymerase with an appropriate primer.

The following are non-limiting examples of polynucleotides: a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications included in thisdefinition are caps, substitution of one or more of the naturallyoccurring nucleotides with an analog, and introduction of means forattaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support. Preferably, thepolynucleotide is DNA. As used herein, “DNA” includes not only bases A,T, C, and G, but also includes any of their analogs or modified forms ofthese bases, such as methylated nucleotides, internucleotidemodifications such as uncharged linkages and thioates, use of sugaranalogs, and modified and/or alternative backbone structures, such aspolyamides.

A polynucleotide or polynucleotide region has a certain percentage (forexample, 80%, 85%, 90%, or 95%) of “sequence identity” to anothersequence means that, when aligned, that percentage of bases are the samein comparing the two sequences. This alignment and the percent homologyor sequence identity can be determined using software programs known inthe art, for example those described in Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987) Supplement 30, section7.7.18, Table 7.7.1. A preferred alignment program is ALIGN Plus(Scientific and Educational Software, Pennsylvania), preferably usingdefault parameters.

“Under transcriptional control” is a term well-understood in the art andindicates that transcription of a polynucleotide sequence, usually a DNAsequence, depends on its being operably (operatively) linked to anelement which contributes to the initiation of, or promotes,transcription. “Operably linked” refers to a juxtaposition wherein theelements are in an arrangement allowing them to function.

“Replication” and “propagation” are used interchangeably and refer tothe ability of a polynucleotide construct of the invention to reproduce,or proliferate. This term is well understood in the art. For purposes ofthis invention, replication involves production of adenovirus proteinsand is generally directed to reproduction of adenovirus. Replication canbe measured using assays standard in the art and described herein, suchas a burst assay, plaque assay, or a one-step growth curve assay.

As used herein, “cytotoxicity” is a term well understood in the art andrefers to a state in which a cell's usual biochemical or biologicalactivities are compromised (i.e., inhibited). These activities include,but are not limited to, metabolism; cellular replication; DNAreplication; transcription; translation; uptake of molecules.“Cytotoxicity” includes cell death and/or cytolysis. Assays are known inthe art which indicate cytotoxicity, such as dye exclusion,.sup.3H-thymidine uptake, and plaque assays.

The term “selective cytotoxicity”, as used herein, refers to thecytotoxicity conferred by an adenovirus vector of the present inventionon a cell which allows or induces a cell status-specific TRE to function(a target cell) when compared to the cytotoxicity conferred by anadenoviral vector of the present invention on a cell which does notallow a cell status-specific TRE to function (a non-target cell). Suchcytotoxicity may be measured, for example, by plaque assays, byreduction or stabilization in size of a tumor comprising target cells,or the reduction or stabilization of serum levels of a markercharacteristic of the tumor cells, or a tissue-specific marker, e.g., acancer marker, such as prostate specific antigen.

In the context of adenovirus, a “heterologous polynucleotide” or“heterologous, gene” or “transgene” is any polynucleotide or gene thatis not present in wild-type adenovirus. Preferably, the transgene willalso not be expressed or present in the target cell prior tointroduction by the adenovirus vector. Examples of preferred transgenesare provided below.

In the context of adenovirus, a “heterologous” promoter or enhancer isone which is not associated with or derived from an adenovirus gene.

In the context of adenovirus, an “endogenous” promoter, enhancer, or TREis native to or derived from adenovirus.

In the context of a cell status-specific TRE, a “heterologous” promoteror enhancer is one which is not normally associated in a cell with orderived from a cell status-specific TRE. Examples of a heterologouspromoter or enhancer are the albumin promoter or enhancer and otherviral promoters and enhancers, such as SV40, or cell type-specific TREssuch as a prostate-specific TRE.

A “cell type-specific TRE” is preferentially functional in a specifictype of cell relative to other types of cells. In contrast to cellstatus, “cell type” is a reflection of a differentiation state of a cellwhich is irreversible. For example, a prostate-specific antigen isexpressed in prostate cells, but is not substantially expressed in othercell types such as hepatocytes, astrocytes, cardiocytes, lymphocytes,etc. Generally, a cell type-specific TRE is active in only one celltype. When a cell type-specific TRE is active in more than one celltype, its activity is restricted to a limited number of cell types,i.e., it is not active in all cell types. A cell type-specific TRE mayor may not be tumor cell specific.

“Suppressing” tumor growth indicates a growth state that is curtailedwhen compared to growth without contact with, i.e., transfection by, anadenoviral vector described herein. Tumor cell growth can be assessed byany means known in the art, including, but not limited to, measuringtumor size, determining whether tumor cells are proliferating using a.sup.3H-thymidine incorporation assay, or counting tumor cells.“Suppressing” tumor cell growth means any or all of the followingstates: slowing, delaying, and stopping tumor growth, as well as tumorshrinkage.

As used herein, the terms “neoplastic cells”, “neoplasia”, “tumor”,“tumor cells”, “cancer” and “cancer cells”, (used interchangeably) referto cells which exhibit relatively autonomous growth, so that theyexhibit an aberrant growth phenotype characterized by a significant lossof control of cell proliferation (i.e., de-regulated cell division).Neoplastic cells can be malignant or benign.

A “host cell” includes an individual cell or cell culture which can beor has been a recipient of an adenoviral vector(s) of this invention.Host cells include progeny of a single host cell, and the progeny maynot necessarily be completely identical (in morphology or in total DNAcomplement) to the original parent cell due to natural, accidental, ordeliberate mutation and/or change. A host cell includes cellstransfected or infected in vivo or in vitro with an adenoviral vector ofthis invention.

“Replication” and “propagation” are used interchangeably and refer tothe ability of an adenovirus vector of the invention to reproduce orproliferate. These terms are well understood in the art. For purposes ofthis invention, replication involves production of adenovirus proteinsand is generally directed to reproduction of adenovirus. Replication canbe measured using assays standard in the art and described herein, suchas a burst assay or plaque assay. “Replication” and “propagation”include any activity directly or indirectly involved in the process ofvirus manufacture, including, but not limited to, viral gene expression;production of viral proteins, nucleic acids or other components;packaging of viral components into complete viruses; and cell lysis.

An “ADP coding sequence” is a polynucleotide that encodes ADP or afunctional fragment thereof. In the context of ADP, a “functionalfragment” of ADP is one that exhibits cytotoxic activity, especiallycell lysis, with respect to adenoviral replication. Ways to measurecytotoxic activity are known in the art and are described herein.

A polynucleotide that “encodes” an ADP polypeptide is one that can betranscribed and/or translated to produce an ADP polypeptide or afragment thereof. The anti-sense strand of such a polynucleotide is alsosaid to encode the sequence.

An “ADP polypeptide” is a polypeptide containing at least a portion, orregion, of the amino acid sequence of an ADP (see, for example, SEQ IDNO:5), and which displays a function associated with ADP, particularlycytotoxicity, more particularly, cell lysis. As discussed herein, thesefunctions can be measured using techniques known in the art. It isunderstood that certain sequence variations may be used, due to, forexample, conservative amino acid substitutions, which may provide ADPpolypeptides.

A polynucleotide sequence that is “depicted in” a SEQ ID NO means thatthe sequence is present as an identical contiguous sequence in the SEQID NO. The term encompasses portions, or regions of the SEQ ID NO aswell as the entire sequence contained within the SEQ ID NO.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals, sportanimals, rodents, primates, and pets.

An “effective amount” is an amount sufficient to effect beneficial ordesired results, which may include clinical results. An effective amountcan be administered in one or more administrations. For purposes of thisinvention, an effective amount of an adenoviral vector is an amount thatis sufficient to palliate, ameliorate, stabilize, reverse, slow or delaythe progression of the disease state.

Adenoviral Vectors Comprising a Cell Status-Specific TRE

The present invention provides adenoviral vector constructs whichcomprise an adenovirus gene under transcriptional control of a cellstatus-specific TRE. Preferably, the adenovirus gene contributes tocytotoxicity (whether direct and/or indirect), more preferably one thatcontributes to or causes cell death, even more preferably is essentialfor advenoviral replication. Examples of a gene that contributes tocytotoxicity include, but are not limited to, adenovirus death protein(ADP; discussed below). When the adenovirus vector(s) is selectively(i.e., preferentially) replication competent for propagation in targetcells, i.e., cells which permit or induce a cell-status TRE to function,these cells will be preferentially killed upon adenoviral proliferation.Once the target cells are destroyed due to selective cytotoxic and/orcytolytic replication, the adenovirus vector replication issignificantly reduced, thus lessening the probability of runawayinfection and undesirable bystander effects. In vitro cultures may beretained to monitor the mixture (such as, for example, a biopsy or otherappropriate biological sample) for occurrence (i.e., presence) and/orrecurrence of the target cell, e.g., a neoplastic cell or otherundesired cell. To further ensure cytotoxicity, one or more transgeneshaving a cytotoxic effect may also be present and under selectivetranscriptional control. In this embodiment, one may provide higherconfidence that the target cells will be destroyed. Additionally, oralternatively, an adenovirus gene that contributes to cytotoxicityand/or cell death (such as ADP) may be included in the adenoviralvector, either free of, or under, selective transcriptional control.

Cell status-specific TREs for use in the adenoviral vectors of thepresent invention can be derived from any species, preferably a mammal.A number of genes have been described which are expressed in responseto, or in association with, a cell status. Any of these cellstatus-associated genes may be used to generate a cell status-specificTRE.

An example of a cell status is cell cycle. An exemplary gene whoseexpression is associated with cell cycle is E2F-1, a ubiquitouslyexpressed, growth-regulated gene, which exhibits peak transcriptionalactivity in S phase. Johnson et al. (1994) Genes Dev. 8:1514-1525. TheRB protein, as well as other members of the RB family, form specificcomplexes with E2F-1, thereby inhibiting its ability to activatetranscription. Thus, E2F-1-responsive promoters are down-regulated byRB. Many tumor cells have disrupted RB function, which can lead tode-repression of E2F-1-responsive promoters, and, in turn, de-regulatedcell division.

Accordingly, in one embodiment, the invention provides an adenoviralvector in which an adenoviral gene (preferably a gene necessary forreplication) is under transcriptional control of a cell status-specificTRE, wherein the cell status-specific TRE comprises a cellcycle-activated, or cell-cycle specific, TRE. In one embodiment, thecell cycle-activated TRE is an E2F1 TRE. In one embodiment, this TREcomprises the sequence depicted in FIG. 3 and SEQ ID NO:2.

Another group of genes which are regulated by cell status are thosewhose expression is increased in response to hypoxic conditions. Bunnand Poyton (1996) Physiol. Rev. 76:839-885; Dachs and Stratford (1996)Br. J. Cancer 74:5126-5132; Guillemin and Krasnow (1997) Cell 89:9-12.Many tumors have insufficient blood supply, due in part to the fact thattumor cells typically grow faster than the endothelial cells that makeup the blood vessels, resulting in areas of hypoxia in the tumor.Folkman (1989) J. Natl. Cancer Inst. 82:4-6; and Kallinowski (1996) TheCancer J. 9:3740. An important mediator of hypoxic responses is thetranscriptional complex HIF-1, or hypoxia inducible factor-1, whichinteracts with a hypoxia-responsive element (HRE) in the regulatoryregions of several genes, including vascular endothelial growth factor,and several genes encoding glycolytic enzymes, including enolase-1.Murine HRE sequences have been identified and characterized. Firth etal. (1994) Proc. Natl. Acad. Sci. USA 91:6496-6500. An HRE from a ratenolase-1 promoter is described in Jiang et al. (1997) Cancer Res.57:5328-5335. An HRE from a rat enolase-1 promoter is depicted in FIG. 2and given as SEQ ID NO:1.

Accordingly, in one embodiment, an adenovirus vector comprises anadenovirus gene, preferably an adenoviral gene essential forreplication, under transcriptional control of a cell status-specific TREcomprising an HRE. In one embodiment, the cell status-specific TREcomprises the HRE depicted in FIG. 2 and SEQ ID NO:1.

Other cell status-specific TREs include heat-inducible (i.e., heatshock) promoters, and promoters responsive to radiation exposure,including ionizing radiation and UV radiation. For example, the promoterregion of the early growth response-1 (Egr-1) gene contains anelement(s) inducible by ionizing radiation. Hallahan et al. (1995) Nat.Med. 1:786-791; and Tsai-Morris et al. (1988) Nucl. Acids. Res.16:8835-8846. Heat-inducible promoters, including heat-inducibleelements, have been described. See, for example Welsh (1990) in “StressProteins in Biology and Medicine”, Morimoto, Tisseres, and Georgopoulos,eds. Cold Spring Harbor Laboratory Press; and Perisic et al. (1989) Cell59:797-806. Accordingly, in some embodiments, the cell status-specificTRE comprises an element(s) responsive to ionizing radiation. In oneembodiment, this TRE comprises a 5′ flanking sequence of an Egr-1 gene.In other embodiments, the cell status-specific TRE comprises a heatshock responsive, or heat-inducible, element.

A cell status-specific TRE can also comprise multimers. For example, anHRE can comprise a tandem series of at least two, at least three, atleast four, or at least five hypoxia-responsive elements. Thesemultimers may also contain heterologous promoter and/or enhancersequences.

A cell status-specific TRE may or may not lack a silencer. The presenceof a silencer (i.e., a negative regulatory element) may assist inshutting off transcription (and thus replication) in non-permissivecells (i.e., cell in a normal cell state). Thus, presence of a silencermay confer enhanced cell status-specific replication by more effectivelypreventing adenoviral vector replication in non-target cells.Alternatively, lack of a silencer may assist in effecting replication intarget cells, thus conferring enhanced cell status-specific replicationdue to more effective replication in target cells.

In other embodiments, the adenoviral vector comprises an adenoviral geneessential for adenoviral replication under control of a first cellstatus-specific TRE, and a second adenoviral gene essential foradenoviral replication under control of a second cell status-specificTRE. The first and the second cell status-specific TREs may or may notbe identical, and may or may not be substantially identical to oneanother. By “substantially identical” is meant a requisite degree ofsequence identity between the two TREs. The degree of sequence identitybetween these TREs is at least about 80%, preferably at least about 85%,more preferably at least about 90%, even more preferably at least about95%, even more preferably at least about 98%, and most preferably 100%.Sequence identity can be determined by a sequence comparison using,i.e., sequence alignment programs that are known in the art, such asthose described in Current Protocols in Molecular Biology (F. M. Ausubelet al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1 Apreferred alignment program is ALIGN Plus (Scientific and EducationalSoftware, Pennsylvania), preferably using default parameters.Alternatively, hybridization under stringent conditions can alsoindicate degree of sequence identity. Stringent conditions are known inthe art; an example of a stringent condition is 80.degree. C.; (orhigher temperature) and 6×SSC (or less concentrated SSC). Otherhybridization conditions and parameters (in order of increasingstringency) are: incubation temperatures of 25.degree. C., 37.degree.C., 50.degree. C., and 68.degree. C.; buffer concentrations of 10×SSC,6×SSC, 1×SSC, 0.1×SSC (where 1×SSC is 0.15 M NaCl and 15 mM citratebuffer) and their equivalents using other buffer systems; formamideconcentrations of 0%, 25%, 50%, and 75%; incubation times from about 24hours about 5 minutes; 1, 2, or more washing steps; wash incubationtimes of 1, 2, or 15 minutes; and wash solutions of 6×SSC, 1×SSC,0.1×SSC, or deionized water.

Adenoviral constructs in which the first and second cell status-specificTREs are identical or substantially identical, particularly if theseTREs control transcription of early genes (such as E1A and E1B), maydisplay an instability which may be desirable in certain contexts, suchas when an automatic “self-destruction” property can shut down thevirus, thereby controlling the degree of propagation. Accordingly, insome embodiments, the first and second cell status-specific TRE, or thefirst and second TRE (one of which is a cell-status-specific TRE) aresufficiently identical to confer instability when compared to two TREswhich are less identical with respect to each other (i.e., have moresequence divergence or dissimilarity). Preferred embodiments are thosein which the two TREs control E1A and E1B respectively. “Instability”means that the structural integrity of the adenoviral vectors is notpreserved as the virus replicates in cells, and can be measured usingstandard methods in the art, such as Southern analysis. In otherembodiments, the first and second TREs are sufficiently divergent and/orplaced in the vector such that the vector is stable (i.e., thestructural integrity of the adenoviral vector is preserved).

In other embodiments, the adenoviral vector comprises an adenoviral geneessential for adenoviral replication under control of a first cellstatus-specific TRE, and a transgene under control of a second cellstatus-specific TRE. The first and the second cell status-specific TREsmay or may not be substantially identical to one another.

In some embodiments, a cell status-specific TRE can be juxtaposed withanother TRE, such as a different cell status-specific TRE, or,alternatively, a cell type-specific TRE. “Juxtaposed” means a cellstatus-specific TRE and the second TRE transcriptionally control thesame gene, or at least are proximate with respect to the same gene. Forthese embodiments, the cell status-specific TRE and the second TRE maybe in any of a number of configurations, including, but not limited to,(a) next to each other (i.e., abutting); (b) both 5′ to the gene that istranscriptionally controlled (i.e., may have intervening sequencesbetween them); (c) one TRE 5′ and the other TRE 3′ to the gene. Forexample, as described in Example 1 and shown in FIG. 1, a celltype-specific TRE can be juxtaposed with a cell status-specific TRE tocontrol transcription of an operably linked adenoviral gene. Such“composite” TREs can be used to confer both cell status- and celltype-specific expression of an operably linked polynucleotide, and thusmay confer significantly greater specificity and/or efficacy. Examplesof cell type-specific TREs are provided below. Alternatively,“composite” TREs can be used to confer different, and possiblysynergistic, cell status specificity. For example, a composite TRE couldconfer specificity to hypoxia and heat shock.

Example 1 provides a description of an adenovirus construct in which acomposite TRE upstream of E1A consisting of an HRE and aprostate-specific TRE, PSA-TRE (which consists of enhancer sequences−5322 to −3738 fused to PSA promoter sequence −541 to +12; see U.S. Pat.Nos. 5,871,726; 5,648,478). Accordingly, in some embodiments, theinvention provides an adenovirus vector comprising an adenovirus geneessential for replication, preferably an early gene, preferably E1A orE1B, under transcriptional control of a TRE comprising an HRE(preferably comprising or consisting of the 67-base fragment depicted inSEQ ID NO:1) and a prostate cell specific TRE, preferably comprising aPSA enhancer (preferably −5322 to −3738; or about 503 to about 2086 ofSEQ ID NO:3 (bases about 503 to about 2086 of FIG. 4), and a promoter,preferably comprising a PSA enhancer and a PSA promoter (about 5285 toabout 5836 of SEQ ID NO:3).

As is readily appreciated by one skilled in the art, a cellstatus-specific TRE is a polynucleotide sequence, and, as such, canexhibit function over a variety of sequence permutations. Methods ofnucleotide substitution, addition, and deletion are known in the art,and readily available functional assays (such as the CAT or luciferasereporter gene assay) allow one of ordinary skill to determine whether asequence variant exhibits requisite cell status-specific transcriptionfunction. Hence, the invention also includes functionally-preservedvariants of the nucleic acid sequences disclosed herein, which includenucleic acid substitutions, additions, and/or deletions. While notwishing to be bound by a single theory, the inventors note that it ispossible that certain modifications will result in modulated resultantexpression levels, including enhanced expression levels. Achievement ofmodulated resultant expression levels, preferably enhanced expressionlevels, may be especially desirable in the case of certain, moreaggressive forms of cancer, or when a more rapid and/or aggressivepattern of cell killing is warranted (due to an immunocompromisedcondition of the individual, for example).

As an example of how cell status-specific TRE activity can bedetermined, a polynucleotide sequence or set of such sequences can begenerated using methods known in the art, such as chemical synthesis,site-directed mutagenesis, PCR, and/or recombinant methods. Thesequence(s) to be tested is inserted into a vector containing anappropriate reporter gene, including, but not limited to,chloramphenicol acetyl transferase (CAT), beta.-galactosidase (encodedby the lacZ gene), luciferase (encoded by the luc gene), greenfluorescent protein, alkaline phosphatase, and horse radish peroxidase.Such vectors and assays are readily available, from, inter alia,commercial sources. Plasmids thus constructed are transfected into asuitable host cell to test for expression of the reporter gene ascontrolled by the putative cell status-specific TRE using transfectionmethods known in the art, such as calcium phosphate precipitation,electroporation, liposomes (lipofection), and DEAE-dextran. Suitablehost cells include any cell type, including but not limited to, Hep3B,Hep G2, HuH7, HuH1/C12, LNCaP, HBL-100, Chang liver cells, MCF-7, HLF,HLE, 3T3, HUVEC, and HeLa. Host cells transfected with the TRE-reportergene construct to be tested are subjected to conditions which result ina change in cell status (for example, one which result in an aberrantphysiological state). The same cells not subjected to these conditions,i.e., which are under normal physiological conditions and therefore in anormal physiological state, serve as controls. Results are obtained bymeasuring the level of expression of the reporter gene using standardassays. Comparison of expression between cells in a particular state andcontrol indicates presence or absence of transcriptional activation.“Transcriptional activation” has been defined above.

Comparisons between or among various cell status-specific TREs can beassessed, for example, by measuring and comparing levels of expressionwithin a single cell line under normal and aberrant physiologicalconditions. These comparisons may also be made by measuring andcomparing levels of expression within a single cell line subjected toreversible environmental conditions (such as heat) and cells notsubjected to such conditions. It is understood that absolutetranscriptional activity of an cell status-specific TRE will depend onseveral factors, such as the nature of the target cell, delivery modeand form of the cell status-specific TRE, and the coding sequence thatis to be selectively transcriptionally activated. To compensate forvarious plasmid sizes used, activities can be expressed as relativeactivity per mole of transfected plasmid. Alternatively, the level oftranscription (i.e., mRNA) can be measured using standard Northernanalysis and hybridization techniques. Levels of transfection (i.e.,transfection efficiencies) are measured by co-transfecting a plasmidencoding a different reporter gene under control of a different TRE,such as the cytomegalovirus (CMV) immediate early promoter. Thisanalysis can also indicate negative regulatory regions, i.e., silencers.

As an example of how hypoxia induction can be measured, one can use anassay such as that described in Jiang et al. (1997) Cancer Research57:5328-5335 or Dachs et al. (1997) Nature Med. 3:515-520. For example,a construct comprising a putative HRE, or multiple tandem copiesthereof, together with a minimal promoter element, operably linked andcontrolling transcription of a polynucleotide which encodes a proteinwhich is detectable or can be used to give a detectable signal, isintroduced into host cells. The host cells are then subjected toconditions of normoxia (e.g., 20% O.sub.2), and varying degrees ofhypoxia, such as 5%, 2%, 1%, 0.1%, or less, O.sub.2. The expressionproduct of the operably linked polynucleotide (reporter gene) is thenmeasured.

Alternatively a putative cell status-specific TRE can be assessed forits ability to confer adenoviral replication preference for cellsexhibiting the requisite physiological state, such as heat or ionizingradiation. For this assay, constructs containing an adenovirus geneessential to replication operably linked to a putative cellstatus-specific TRE are transfected into cells which exhibit therequisite physiological state. Viral replication in those cells iscompared, for example, to viral replication by the construct in cellsunder normal physiological conditions (i.e., not exhibiting therequisite physiological state).

Any of the various serotypes of adenovirus can be used, such as Ad2,Ad5, Ad12 and Ad40. For purposes of illustration, serotype Ad5 will beexemplified herein.

When a cell status-specific TRE is used with an adenovirus gene that isessential for propagation replication competence is preferentiallyachievable in the target cell expressing cell status. Preferably, thegene is an early gene, such as E1A, E1B, E2, or E4. (E3 is not essentialfor viral replication). More preferably, the early gene under cellstatus-TRE control is E1A and/or E1B. More than one early gene can beplaced under control of an cell status-specific TRE. Example 1 providesa more detailed description of such constructs.

The E1A gene is expressed immediately after viral infection (0-2 hours)and before any other viral genes. E1A protein acts as a trans-actingpositive-acting transcriptional regulatory factor, and is required forthe expression of the other early viral genes E1B, E2, E3, E4, and thepromoter-proximal major late genes. Despite the nomenclature, thepromoter proximal genes driven by the major late promoter are expressedduring early times after Ad5 infection. Flint (1982) Biochem. Biophys.Acta 651:175-208; Flint (1986) Advances Virus Research 31:169-228; Grand(1987) Biochem. J. 241:25-38. In the absence of a functional E1A gene,viral infection does not proceed, because the gene products necessaryfor viral DNA replication are not produced. Nevins (1989) Adv. VirusRes. 31:35-81. The transcription start site of Ad5 E1A is at 498 and theATG start site of the E1A protein is at 560 in the virus genome.

The E1B protein functions in trans and is necessary for transport oflate mRNA from the nucleus to the cytoplasm. Defects in E1B expressionresult in poor expression of late viral proteins and an inability toshut off host cell protein synthesis. The promoter of E1B has beenimplicated as the defining element of difference in the host range ofAd40 and Ad5: clinically Ad40 is an enterovirus, whereas Ad5 causesacute conjunctivitis. Bailey, Mackay et al. (1993) Virology 193:631;Bailey et al. (1994) Virology 202:695-706). The E1B promoter of Ad5consists of a single high-affinity recognition site for Sp1 and a TATAbox.

The E2 region of adenovirus codes for proteins related to replication ofthe adenoviral genome, including the 72 kDa DNA-binding protein, the 80kD precursor terminal protein and the viral DNA polymerase. The E2region of Ad5 is transcribed in a rightward orientation from twopromoters, termed E2 early and E2 late, mapping at 76.0 and 72.0 mapunits, respectively. While the E2 late promoter is transiently activeduring late stages of infection and is independent of the E1Atransactivator protein, the E2 early promoter is crucial during theearly phases of viral replication.

The E2 late promoter overlaps with the coding sequences of a geneencoded by the counterstrand and is therefore not amenable to geneticmanipulation. However, the E2 early promoter overlaps only for a fewbase pairs with sequences coding for a 33 kD protein on thecounterstrand. Notably, the SpeI restriction site (Ad5 position 27082)is part of the stop codon for the above mentioned 33 kD protein andconveniently separates the major E2 early transcription initiation siteand TATA-binding protein site from the upstream transcription factorbiding sites E2F and ATF. Therefore, insertion of a cell status-TREhaving SpeI ends into the SpeI site in the +-strand would disrupt theendogenous E2 early promoter of Ad5 and should allow cellstatus-restricted expression of E2 transcripts.

The E4 gene has a number of transcription products. The E4 region codesfor two polypeptides which are responsible for stimulating thereplication of viral genomic DNA and for stimulating late geneexpression. The protein products of open reading frames (ORFS) 3 and 6can both perform these functions by binding the 55 kD protein from E1Band heterodimers of E2F-1 and DP-1. The ORF 6 protein requiresinteraction with the E1B 55 kD protein for activity while the ORF 3protein does not. In the absence of functional protein from ORF 3 andORF 6, plaques are produced with an efficiency less than 10.sup.-6 thatof wild type virus. To further restrict viral replication to cellsexhibiting a requisite physiological condition or state, E4 ORFs 1-3 canbe deleted, making viral DNA replication and late gene synthesisdependent on E4 ORF 6 protein. By combining such a mutant with sequencesin which the EIB region is regulated by a cell status-specific TRE, avirus can be obtained in which both the E1B function and E4 function aredependent on a cell status-specific TRE driving E1B.

The major late genes relevant to the subject invention are genes L1, L2,L3, L4, and L5 which encode proteins of the adenovirus virion. All ofthese genes (typically coding for structural proteins) are probablyrequired for adenoviral replication. The late genes are all under thecontrol of the major late promoter (MLP), which is located in Ad5 at+5986 to +6048.

In addition to conferring selective cytotoxic and/or cytolytic activityby virtue of preferential replication competence in cells exhibiting arequisite physiological state (for example, an aberrant physiologicalstate such as low oxygen conditions), the adenovirus vectors of thisinvention can further include a heterologous gene (transgene) under thecontrol of a cell status-specific TRE. In this way, various geneticcapabilities may be introduced into target cells, particularly cancercells. For example, in certain instances, it may be desirable to enhancethe degree and/or rate of cytotoxic activity, due to, for example, therelatively refractory nature or particular aggressiveness of thecancerous target cell. This could be accomplished by coupling the cellstatus-specific replicative cytotoxic activity with cell-specificexpression of, for example, HSV-tk and/or cytosine deaminase (cd), whichrenders cells capable of metabolizing 5-fluorocytosine (5-FC) to thechemotherapeutic agent 5-fluorouracil (5-FU). Using these types oftransgenes may also confer a bystander effect.

Other desirable transgenes that may be introduced via an adenovirusvector(s) include genes encoding cytotoxic proteins, such as the Achains of diphtheria toxin, ricin or abrin (Palmiter et al. (1987) Cell50:435; Maxwell et al. (1987) Mol. Cell. Biol. 7:1576; Behringer et al.(1988) Genes Dev. 2:453; Messing et al. (1992) Neuron 8:507; Piatak etal. (1988) J. Biol. Chem. 263:4937; Lamb et al. (1985) Eur. J. Biochem.148:265; Frankel et al. (1989) Mol. Cell. Biol. 9:415), genes encoding afactor capable of initiating apoptosis, sequences encoding antisensetranscripts or ribozymes, which among other capabilities may be directedto mRNAs encoding proteins essential for proliferation, such asstructural proteins, or transcription factors; viral or other pathogenicproteins, where the pathogen proliferates intracellularly; genes thatencode an engineered cytoplasmic variant of a nuclease (e.g. RNase A) orprotease (e.g. awsin, papain, proteinase K, carboxypeptidase, etc.), orencode the Fas gene, and the like. Other genes of interest includecytokines, antigens, transmembrane proteins, and the like, such as IL-1,-2, -6, -12, GM-CSF, G-CSF, M-CSF, IFN-.alpha., -.beta., -.gamma.,TNF-.alpha., -beta., TGF-.alpha., -.beta., NGF, and the like. Thepositive effector genes could be used in an earlier phase, followed bycytotoxic activity due to replication.

In one embodiment, the adenovirus death protein (ADP), encoded withinthe E3 region, is maintained in the adenovirus vector. The ADP gene,under control of the major late promoter (MLP), appears to code for aprotein (ADP) that is important in expediting host cell lysis. Tollefsonet al. (1996) J. Virol. 70(4):2296; Tollefson et al. (1992) J. Virol.66(6):3633. Thus, adenoviral vectors containing the ADP gene may renderthe adenoviral vector more potent, making possible more effectivetreatment and/or a lower dosage requirement.

Accordingly, the invention provides an adenoviral vector as describedherein that further includes a polynucleotide sequence encoding an ADP.A DNA sequence encoding an ADP and the amino acid sequence of an ADP aredepicted FIG. 9. Briefly, an ADP coding sequence is obtained preferablyfrom Ad2 (since this is the strain in which ADP has been more fullycharacterized) using techniques known in the art, such as PCR.Preferably, the Y leader (which is an important sequence for correctexpression of late genes) is also obtained and ligated to the ADP codingsequence. The ADP coding sequence (with or without the Y leader) canthen be introduced into the adenoviral genome, for example, in the E3region (where the ADP coding sequence will be driven by the MLP). TheADP coding sequence could also be inserted in other locations of theadenovirus genome, such as the E4 region. Alternatively, the ADP codingsequence could be operably linked to a heterologous promoter (with orwithout enhancer(s)), including, but not limited to, another viralpromoter, a cell status-specific TRE such as a hypoxia responsiveelement, or a cell type-specific TRE such as those derived fromcarcinoembryonic antigen (CEA), mucin, and rat probasin genes.

Adenoviral Vectors of the Invention Further Comprising a Cell TypeSpecific Element

In addition to conferring selective cytotoxic and/or cytolytic activityby virtue of preferential replication competence and/or by preferentialtranscription of a gene encoding a cytotoxic factor in cells exhibitinga requisite physiological state, the adenovirus vectors of thisinvention can further include an adenovirus gene and/or a heterologousgene (transgene) under the control of a cell type-specific TRE. In thisway, cytotoxicity is further limited to a particular cell type.

For example, TREs that function preferentially in prostate cellsinclude, but are not limited to, TREs derived from the prostate-specificantigen gene (PSA-TRE) (U.S. Pat. No. 5,648,478), the glandularkallikrein-1 gene (from the human gene, hKLK2-TRE), and the probasingene (PB-TRE) (International Patent Application No. PCT/US98/04132). Allthree of these genes are preferentially expressed in prostate cells andthe expression is androgen-inducible. Generally, expression of genesresponsive to androgen induction requires the presence of an androgenreceptor (AR).

PSA is synthesized exclusively by normal, hyperplastic, and malignantprostatic epithelia; hence, its tissue-specific expression has made itan excellent biomarker for benign prostatic hyperplasia (BPH) andprostatic carcinoma (CaP). Normal serum levels of PSA are typicallybelow 5 ng/ml, with elevated levels indicative of BPH or CaP. Lundwallet al. (1987) FEBS Lett. 214:317; Lundwall (1989) Biochem. Biophys. Res.Comm. 161:1151; and Riegmann et al. (1991) Molec. Endocrin. 5:1921.

The region of the PSA gene that is used to provide cell specificitydependent upon androgens, particular in prostate cells, involvesapproximately 6.0 kilobases. Schuur et al. (1996) J. Biol. Chem.271:7043-7051. An enhancer region of approximately 1.5 kb in humans islocated between nt −5322 and nt −3738, relative to the transcriptionstart site of the PSA gene. The PSA promoter consists of the sequencefrom about nt −540 to nt +12 relative to the transcription start site.Juxtapositioning of these two genetic elements yield a fully functional,minimal prostate-specific enhancer/promoter (PSE) TRE. Other portions ofthe approximately 6.0 kb region of the PSA gene can be used in thepresent invention, as long as requisite functionality is maintained. InExample 1, adenoviral vector CN796 is described which comprises acomposite TRE comprising an HRE and a PSA-TRE, the PSA-TRE comprising aPSA enhancer from −5322 to −3738 fused to a PSA promoter from −541 to+12. This PSA-TRE is derived from adenoviral vector CN706. Rodriguez etal. (1997) Cancer Research 57:2559-2563. Accordingly, in one embodimentan adenoviral vector comprises and adenovirus E1A gene undertranscriptional control of a composite TRE comprising the cellstatus-specific TRE, HRE, and a cell type-specific TRE, a PSA-TRE.

The PSE and PSA TRE used in the present invention are derived fromsequences depicted in FIG. 4 (SEQ ID NO:3). The enhancer element isnucleotides about 503 to about 2086 of FIG. 4 (SEQ ID NO:3). Thepromoter is nucleotides about 5285 to about 5836 of FIG. 4 (SEQ IDNO:3). Accordingly, in some embodiments, the composite TRE comprises anHRE comprising SEQ ID NO:1 and a PSA-TRE comprises nucleotides about 503to about 2086 of SEQ ID NO:3. In other embodiments, the composite TREcomprises an HRE comprising SEQ ID NO:1 and a PSA-TRE comprisesnucleotides about 503 to about 2086 of SEQ ID NO:3 and nucleotides about5285 to about 5836 of SEQ ID NO:3. As described above, these composite(HRE/PSA) TREs may be operably linked to an adenovirus gene essentialfor replication, especially an early gene such as E1A or E1B. Example 1describes such a construct.

In the present invention, replication-competent adenovirus vectorscomprising a cell status-specific TRE and a cell type-specific TRE mayemploy cell type-specific TREs that are preferentially functional inparticular tumor cells. Non-limiting examples of tumor cell-specificTREs, and non-limiting examples of respective potential target cells,include TREs from the following genes: .alpha.-fetoprotein (AFP) (livercancer), mucin-like glycoprotein DF3 (MUC1) (breast carcinoma),carcinoembryonic antigen (CEA) (colorectal, gastric, pancreatic, breast,and lung cancers), plasminogen activator urokinase (uPA) and itsreceptor gene (breast, colon, and liver cancers), HER-2/neu(c-erbB2/neu) (breast, ovarian, stomach, and lung cancers).

Other cell type-specific TREs may be derived from the followingexemplary genes (cell type in which the TREs are specifically functionalare in parentheses): vascular endothelial growth factor receptor(endothelium), albumin (liver), factor VII (liver), fatty acid synthase(liver), von Willebrand factor (brain endothelium), alpha-actin andmyosin heavy chain (both in smooth muscle), synthetase I (smallintestine), Na—K—Cl transporter (kidney). Additional cell type-specificTREs are known in the art.

AFP is an oncofetal protein, the expression of which is primarilyrestricted to developing tissues of endodermal origin (yolk sac, fetalliver, and gut), although the level of its expression varies greatlydepending on the tissue and the developmental stage. AFP is of clinicalinterest because the serum concentration of AFP is elevated in amajority of hepatoma patients, with high levels of AFP found in patientswith advanced disease. The serum AFP levels in patients appear to beregulated by AFP expression in hepatocellular carcinoma but not insurrounding normal liver. Thus, the AFP gene appears to be regulated tohepatoma cell-specific expression.

Cell type-specific TREs from the AFP gene have been identified. Forexample, the cloning and characterization of human AFP-specific enhanceractivity is described in Watanabe et al. (1987) J. Biol. Chem.262:4812-4818. The entire 5′ AFP flanking region (containing thepromoter, putative silencer, and enhancer elements) is contained withinapproximately 5 kb upstream from the transcription start site.

The AFP enhancer region in human is located between about nt −3954 andabout nt −3335, relative to the transcription start site of the AFPgene. The human AFP promoter encompasses a region from about nt −174 toabout nt +29. Juxtapositioning of these two genetic elements yields afully functional AFP-TRE. Ido et al. (1995) describe a 259 bp promoterfragment (nt −230 to nt +29) that is specific for HCC. Cancer Res.55:3105-3109. The AFP enhancer contains two regions, denoted A and B,located between nt −3954 and nt −3335 relative to the transcriptionstart site. The promoter region contains typical TATA and CAAT boxes.Preferably, the AFP-TRE contains at least one enhancer region. Morepreferably, the AFP-TRE contains both enhancer regions.

Suitable target cells for adenoviral vectors containing AFP-TREs are anycell type that allow an AFP-TRE to function. Preferred are cells thatexpress, or produce, AFP, including, but not limited to, tumor cellsexpressing AFP. Examples of such cells are hepatocellular carcinomacells, gonadal and other germ cell tumors (especially endodermal sinustumors), brain tumor cells, ovarian tumor cells, acinar cell carcinomaof the pancreas (Kawamoto et al. (1992) Hepatogastroenterology39:282-286), primary gall bladder tumor (Katsuragi et al. (1989) RinskoHoshasen 34:371-374), uterine endometrial adenocarcinoma cells (Koyamaet al. (1996) Jpn. J. Cancer Res. 87:612-617), and any metastases of theforegoing (which can occur in lung, adrenal gland, bone marrow, and/orspleen). In some cases, metastatic disease to the liver from certainpancreatic and stomach cancers produce AFP. Especially preferred arehepatocellular carcinoma cells and any of their metastases. AFPproduction can be measured using assays standard in the art, such asRIA, ELISA or Western blots (immunoassays) to determine levels of AFPprotein production or Northern blots to determine levels of AFP MRNAproduction. Alternatively, such cells can be identified and/orcharacterized by their ability to activate transcriptionally an AFP-TRE(i.e., allow an AFP-TRE to function).

The protein urokinase plasminogen activator (uPA) and its cell surfacereceptor, urokinase plasminogen activator receptor (uPAR), are expressedin many of the most frequently occurring neoplasia and appear torepresent important proteins in cancer metastasis. Both proteins areimplicated in breast, colon, prostate, liver, renal, lung and ovariancancer. Transcriptional regulatory elements that regulate uPA and uPARtranscription have been extensively studied. Riccio et al. (1985)Nucleic Acids Res. 13:2759-2771; Cannio et al. (1991) Nucleic Acids Res.19:2303-2308.

CEA is a 180,000-Dalton glycoprotein tumor-associated antigen present onendodermally-derived neoplasia of the gastrointestinal tract, such ascolorectal, gastric (stomach) and pancreatic cancer, as well as otheradenocarcinomas such as breast and lung cancers. CEA is of clinicalinterest because circulating CEA can be detected in the great majorityof patients with CEA-positive tumors. In lung cancer, about 50% of totalcases have circulating CEA, with high concentrations of CEA (greaterthan 20 ng/ml) often detected in adenocarcinomas. Approximately 50% ofpatients with gastric carcinoma are serologically positive for CEA.

The 5′ upstream flanking sequence of the CEA gene has been shown toconfer cell-specific activity. The CEA promoter region, approximatelythe first 424 nucleotides upstream of the translational start site inthe 5′ flanking region of the gene, was shown to confer cell-specificactivity when the region provided higher promoter activity inCEA-producing cells than in non-producing HeLa cells. Schrewe et al.(1990) Mol. Cell. Biol. 10:2738-2748. In addition, cell-specificenhancer regions have been found. WO/95/14100. The entire 5′CEA flankingregion (containing the promoter, putative silencer, and enhancerelements) appears to be contained within approximately 14.5 kb upstreamfrom the transcription start site. Richards et al. (1995); WO 95/14100.Further characterization of the 5′ flanking region of the CEA gene byRichards et al. (1995) indicated two upstream regions, −13.6 to −10.7 kbor −6.1 to −4.0 kb, when linked to the multimerized promoter resulted inhigh-level and selective expression of a reporter construct inCEA-producing LoVo and SW1463 cells. Richards et al. (1995) alsolocalized the promoter region to nt −90 and nt +69 relative to thetranscriptional start site, with region nt 41 to nt −18 as essential forexpression. WO95/14100 describes a series of 5′ flanking CEA fragmentswhich confer cell-specific activity, such as about nt −299 to about nt+69; about nt −90 to about nt +69; nt −14,500 to nt −10,600; nt −13,600to nt −10,600, nt −6100 to nt −3800. In addition, cell specifictranscription activity is conferred on an operably linked gene by theCEA fragment from nt 402 to nt +69, depicted in (SEQ ID NO:6). AnyCEA-TREs used in the present invention are derived from mammalian cells,including but not limited to, human cells. Thus, any of the CEA-TREs maybe used in the invention as long as requisite desired functionality isdisplayed in the adenovirus vector. The cloning and characterization ofCEA sequences have been described in the literature and are thus madeavailable for practice of this invention and need not be described indetail herein.

The protein product of the MUC1 gene (known as mucin or MUC1 protein;episialin; polymorphic epithelial mucin or PEM; EMA; DF3 antigen; NPGP;PAS-O; or CA15.3 antigen) is normally expressed mainly at the apicalsurface of epithelial cells lining the glands or ducts of the stomach,pancreas, lungs, trachea, kidney, uterus, salivary glands, and mammaryglands. Zotter et al. (1988) Cancer Rev. 11-12: 55-101; and Girling etal. (1989) Int. J. Cancer 43:1072-1076. However, mucin is overexpressedin 75-90% of human breast carcinomas. Kufe et al. (1984) Hybridoma3:223-232. For reviews, see Hilkens (1988) Cancer Rev. 11-12: 25-54; andTaylor-Papadimitriou, et al. (1990) J. Nucl. Med. Allied Sci.34:144-150. Mucin protein expression correlates with the degree ofbreast tumor differentiation. Lundy et al. (1985) Breast Cancer Res.Treat. 5:269-276. This overexpression appears to be controlled at thetranscriptional level.

Overexpression of the MUC1 gene in human breast carcinoma cells MCF-7and ZR-75-1 appears to be regulated at the transcriptional level. Kufeet al. (1984); Kovarik (1993) J. Biol. Chem. 268:9917-9926; and Abe etal. (1990) J. Cell. Physiol. 143:226-231. The regulatory sequences ofthe MUC1 gene have been cloned, including the approximately 0.9 kbupstream of the transcription start site which contains a TRE thatappears to be involved in cell-specific transcription. Abe et al. (1993)Proc. Natl. Acad. Sci. USA 90:282-286; Kovarik et al. (1993); andKovarik et al. (1996) J. Biol. Chem. 271:18140-18147.

Any MUC1-TREs used in the present invention are derived from mammaliancells, including but not limited to, human cells. Preferably, theMUC1-TRE is human. In one embodiment, the MUC1-TRE may contain theentire 0.9 kb 5′ flanking sequence of the MUC1 gene. In otherembodiments, the MUC1-TREs comprise the following sequences (relative tothe transcription start site of the MUC1 gene): about nt −725 to aboutnt +31, nt −743 to about nt +33, nt −750 to about nt +33, and nt −598 toabout nt +485 (operably-linked to a promoter).

The c-erbB2/neu gene (HER-2/neu or HER) is a transforming gene thatencodes a 185 kD epidermal growth factor receptor-related transmembraneglycoprotein. In humans, the c-erbB2/neu protein is expressed duringfetal development, however, in adults, the protein is weakly detectable(by immunohistochemistry) in the epithelium of many normal tissues.Amplification and/or over-expression of the c-erbB2/neu gene has beenassociated with many human cancers, including breast, ovarian, uterine,prostate, stomach and lung cancers. The clinical consequences of thec-erbB2/neu protein over-expression have been best studied in breast andovarian cancer. c-erbB2/neu protein over-expression occurs in 20 to 40%of intraductal carcinomas of the breast and 30% of ovarian cancers, andis associated with a poor prognosis in subcategories of both diseases.Human, rat and mouse c-erbB2/neu TREs have been identified and shown toconfer c-erbB2/neu expressing cell specific activity. Tal et al. (1987)Mol. Cell. BioL. 7:2597-2601; Hudson et al. (1990) J. Biol. Chem.265:4389-4393; Grooteclaes et al. (1994) Cancer Res. 54:4193-4199; Ishiiet al. (1987) Proc. Natl. Acad. Sci. USA 84:4374-4378; Scott et al.(1994) J. Biol. Chem. 269:19848-19858.

The cell type-specific TREs listed above are provided as non-limitingexamples of TREs that would function in the instant invention.Additional cell-specific TREs are known in the art, as are methods toidentify and test cell specificity of suspected TREs.

Using the Adenoviral Vectors of the Invention

The adenoviral vectors can be used in a variety of forms, including, butnot limited to, naked polynucleotide (usually DNA) constructs;polynucleotide constructs complexed with agents to facilitate entry intocells, such as cationic liposomes or other cationic compounds such aspolylysine; packaged into infectious adenovirus particles (which mayrender the adenoviral vector(s) more immunogenic); packaged into otherparticulate viral forms such as HSV or AAV; complexed with agents (suchas PEG) to enhance or dampen an immune response; complexed with agentsthat facilitate in vivo transfection, such as DOTMA.sup.TM.,DOTAP.sup.TM., and polyamines. Thus, the invention also provides anadenovirus capable of replicating preferentially in cellstatus-producing cells. “Replicating preferentially” means that theadenovirus replicates more in cell exhibiting a requisite physiologicalstate than a cell not exhibiting that state. Preferably, the adenovirusreplicates at least about 2-fold higher, preferably at least about5-fold higher, more preferably at least about 10-fold higher, still morepreferably at least about 50-fold higher, even more preferably at leastabout 100-fold higher, still more preferably at least about 400-fold toabout 500-fold higher, still more preferably at least about 1000-foldhigher, most preferably at least about 1.times.10.sup.6 higher. Mostpreferably, the adenovirus replicates solely in cells exhibiting arequisite physiological state (that is, does not replicate or replicatesat very low levels in cells not exhibiting the requisite physiologicalstate).

If an adenoviral vector is packaged into an adenovirus, the adenovirusitself may also be selected to further enhance targeting. For example,adenovirus fibers mediate primary contact with cellular receptor(s)aiding in tropism. See, e.g., Amberg et al. (1997) Virol. 227:239-244.If a particular subgenus of an adenovirus serotype displayed tropism fora target cell type and/or reduced affinity for non-target cell types,such subgenus (or subgenera) could be used to further increasecell-specificity of cytoxicity and/or cytolysis.

The adenoviral vectors may be delivered to the target cell in a varietyof ways, including, but not limited to, liposomes, general transfectionmethods that are well known in the art (such as calcium phosphateprecipitation or electroporation), direct injection, and intravenousinfusion. The means of delivery will depend in large part on theparticular adenoviral vector (including its form) as well as the typeand location of the target cells (i.e., whether the cells are in vitroor in vivo).

If used as a packaged adenovirus, adenovirus vectors may be administeredin an appropriate physiologically acceptable carrier at a dose of about10.sup.4 to about 10.sup.14. The multiplicity of infection willgenerally be in the range of about 0.001 to 100. If administered as apolynucleotide construct (i.e., not packaged as a virus) about 0.01.mu.g to about 1000 .mu.g of an adenoviral vector can be administered.The adenoviral vector(s) may be administered one or more times,depending upon the intended use and the immune response potential of thehost, and may also be administered as multiple, simultaneous injections.If an immune response is undesirable, the immune response may bediminished by employing a variety of immunosuppressants, so as to permitrepetitive administration, without a strong immune response. If packagedas another viral form, such as HSV, an amount to be administered isbased on standard knowledge about that particular virus (which isreadily obtainable from, for example, published literature) and can bedetermined empirically.

Host Cells Comprising the Adenoviral Vectors of the Invention

The present invention also provides host cells comprising (i.e.,transformed with) the adenoviral vectors described herein. Bothprokaryotic and eukaryotic host cells can be used as long as sequencesrequisite for maintenance in that host, such as appropriate replicationorigin(s), are present. For convenience, selectable markers are alsoprovided. Prokaryotic host cells include bacterial cells, for example,E. coli and mycobacteria. Among eukaryotic host cells are yeast, insect,avian, plant and mammalian. Host systems are known in the art and neednot be described in detail herein.

Compositions of the Invention

The present invention also provides compositions, includingpharmaceutical compositions, containing the adenoviral vectors describedherein. Such compositions (especially pharmaceutical compositions) areuseful for administration in vivo, for example, when measuring thedegree of transduction and/or effectiveness of cell killing in anindividual. Pharmaceutical compositions, comprised an adenoviral vectorof this invention in a pharmaceutically acceptable excipient (generallyan effective amount of the adenoviral vector), are suitable for systemicadministration to individuals in unit dosage forms, sterile parenteralsolutions or suspensions, sterile non-parenteral solutions or oralsolutions or suspensions, oil in water or water in oil emulsions and thelike. Formulations for parenteral and nonparenteral drug deliveryregion, of the amino acid sequence of an ADP (see, for example, SEQ IDNO:5), and are known in the art and are set forth in Remington'sPharmaceutical Sciences, 19th Edition, Mack Publishing (1995).Pharmaceutical compositions also include lyophilized and/orreconstituted forms of the adenoviral vectors (including those packagedas a virus, such as adenovirus) of the invention.

Other compositions are used, and are useful for, detection methodsdescribed herein. For these compositions, the adenoviral vector usuallyis suspended in an appropriate solvent or solution, such as a buffersystem. Such solvent systems are well known in the art.

Kits of the Invention

The present invention also encompasses kits containing an adenoviralvector(s) of this invention. These kits can be used for diagnosticand/or monitoring purposes, preferably monitoring. Procedures usingthese kits can be performed by clinical laboratories, experimentallaboratories, medical practitioners, or private individuals. Kitsembodied by this invention allow someone to detect the presence of cellstatus-producing cells in a suitable biological sample, such as biopsyspecimens.

The kits of the invention comprise an adenoviral vector described hereinin suitable packaging. The kit may optionally provide additionalcomponents that are useful in the procedure, including, but not limitedto, buffers, developing reagents, labels, reacting surfaces, means fordetection, control samples, instructions, and interpretive information.

Preparation of the Adenovirus Vectors of the Invention

The adenovirus vectors of this invention can be prepared usingrecombinant techniques that are standard in the art. Generally, a cellstatus-specific TRE is inserted 5′ to the adenoviral gene of interest,preferably one or more early genes (although late gene(s) may be used).A cell status-specific TRE can be prepared using oligonucleotidesynthesis (if the sequence is known) or recombinant methods (such as PCRand/or restriction enzymes). Convenient restriction sites, either in thenatural adeno-DNA sequence or introduced by methods such asoligonucleotide directed mutagenesis and PCR, provide an insertion sitefor a cell status-specific TRE. Accordingly, convenient restrictionsites for annealing (i.e., inserting) a cell status-specific TRE can beengineered onto the 5′ and 3′ ends of a cell status-specific TRE usingstandard recombinant methods, such as PCR.

Polynucleotides used for making adenoviral vectors of this invention maybe obtained using standard methods in the art, such as chemicalsynthesis, by recombinant methods, and/or by obtaining the desiredsequence(s) from biological sources.

Adenoviral vectors are conveniently prepared by employing two plasmids,one plasmid providing for the left hand region of adenovirus and theother plasmid providing for the right hand region, where the twoplasmids share at least about 500 nt of middle region for homologousrecombination. In this way, each plasmid, as desired, may beindependently manipulated, followed by cotransfection in a competenthost, providing complementing genes as appropriate, or the appropriatetranscription factors for initiation of transcription from a cellstatus-specific TRE for propagation of the adenovirus. Plasmids aregenerally introduced into a suitable host cell such as 293 cells usingappropriate means of transduction, such as cationic liposomes.Alternatively, in vitro ligation of the right and left-hand portions ofthe adenovirus genome can also be used to construct recombinantadenovirus derivative containing all the replication-essential portionsof adenovirus genome. Berkner et al. (1983) Nucleic Acid Research11:6003-6020; Bridge et al. (1989) J. Virol. 63:631-638.

For convenience, plasmids are available that provide the necessaryportions of adenovirus. Plasmid pXC.1 (McKinnon (1982) Gene 19:3342)contains the wild-type left-hand end of Ad5. pBHG10 (Bett et al. (1994)Proc. Natl. Acad. Sci. USA 91:8802-8806; Microbix Biosystems Inc.,Toronto) provides the right-hand end of Ad5, with a deletion in E3. Thedeletion in E3 provides room in the virus to insert a 3 kb cellstatus-TRE without deleting the endogenous enhancer/promoter. Bett etal. (1994). The gene for E3 is located on the opposite strand from E4(r-strand). pBHG11 provides an even larger E3 deletion (an additional0.3 kb is deleted). Bett et al. (1994).

For manipulation of the early genes, the transcription start site of Ad5E1A is at 498 and the ATG start site of the E1A protein is at 560 in thevirus genome. This region can be used for insertion of an cellstatus-specific TRE. A restriction site may be introduced by employingpolymerase chain reaction (PCR), where the primer that is employed maybe limited to the Ad5 genome, or may involve a portion of the plasmidcarrying the Ad5 genomic DNA. For example, where pBR322 is used, theprimers may use the EcoRI site in the pBR322 backbone and the XbaI siteat 1339 of Ad5. By carrying out the PCR in two steps, where overlappingprimers at the center of the region introduce a 30 sequence changeresulting in a unique restriction site, one can provide for insertion ofheterologous TRE at that site.

A similar strategy may also be used for insertion of a heterologous TREto regulate E1B. The E1B promoter of Ad5 consists of a singlehigh-affinity recognition site for Sp1 and a TATA box. This regionextends from 1636 to 1701. By insertion of a heterologous TRE in thisregion, one can provide for target cell-specific transcription of theE1B gene. By employing the left-hand region modified with a heterologousTRE regulating E1A as the template for introducing a heterologous TRE toregulate E1B, the resulting adenovirus vector will be dependent upon thecell status-specific transcription factors for expression of both E1Aand E1B.

Similarly, a cell status-specific TRE can be inserted upstream of the E2gene to make its expression cell status specific. The E2 early promoter,mapping in Ad5 from 27050-27150, consists of a major and a minortranscription initiation site, the latter accounting for about 5% of theE2 transcripts, two non-canonical TATA boxes, two E2F transcriptionfactor binding sites and an ATF transcription factor binding site. For adetailed review of the E2 promoter architecture see Swaminathan et al.,Curr. Topics in Micro. and Imm. (1995) 199 (part 3):177-194.

For E4, one must use the right hand portion of the adenovirus genome.The E4 transcription start site is predominantly at 35609, the TATA boxat 35638 and the first ATG/CTG of ORF 1 is at 35532. Virtanen et al.(1984) J. Virol. 51:822-831. Using any of the above strategies for theother genes, a cell status-specific TRE may be introduced upstream fromthe transcription start site. For the construction of mutants in the E4region, the co-transfection and homologous recombination are performedin W162 cells (Weinberg et al. (1983) Proc. Natl. Acad. Sci.80:5383-5386) which provide E4 proteins in trans to complement defectsin synthesis of these proteins. Alternatively, these constructs can beproduced by in vitro ligation.

Methods Using the Adenovirus Vectors of the Invention

The adenoviral vectors of the invention can be used for a wide varietyof purposes, which will vary with the desired or intended result.Accordingly, the present invention includes methods using the adenoviralvectors described above.

In one embodiment, methods are provided for conferring selectivecytoxicity in target cells (i.e., cells exhibiting a requisitephysiological state which allows a cell status-specific TRE tofunction), generally but not necessarily in an individual (preferablyhuman), comprising contacting the cells with an adenovirus vectordescribed herein, such that the adenovirus vector enters the cell.Cytotoxicity can be measured using standard assays in the art, such asdye exclusion, .sup.3H-thymidine incorporation, and/or lysis.

In another embodiment, methods are provided for propagating anadenovirus specific for mammalian cells which allow a cellstatus-specific TRE to function. These methods entail combining anadenovirus vector with mammalian cells, whereby said adenovirus ispropagated.

The invention further provides methods of suppressing tumor cell growth,generally but not necessarily in an individual (preferably human),comprising contacting a tumor cell with an adenoviral vector of theinvention such that the adenoviral vector enters the tumor cell andexhibits selective cytotoxicity for the tumor cell. Tumor cell growthcan be assessed by any means known in the art, including, but notlimited to, measuring tumor size, determining whether tumor cells areproliferating using a .sup.3H-thymidine incorporation assay, or countingtumor cells.

The invention also includes methods for detecting target cells (i.e.,cells which permit or induce a cell status-specific TRE to function) ina biological sample. These methods are particularly useful formonitoring the clinical and/or physiological condition of an individual(i.e., mammal), whether in an experimental or clinical setting. Forthese methods, cells of a biological sample are contacted with anadenovirus vector, and replication of the adenoviral vector is detected.A suitable biological sample is one in which cells exhibiting arequisite physiological (and/or environmental) state, for example, anaberrant physiological state (such as cells in hypoxic conditions andexhibiting a phenotype characteristic of cells in hypoxic conditions,such as expression of HIF-1) may be or are suspected to be present.Generally, in mammals, a suitable clinical sample is one in whichcancerous cells exhibiting a requisite physiological state, such ascells within a solid tumor which are under hypoxic conditions, aresuspected to be present. Such cells can be obtained, for example, byneedle biopsy or other surgical procedure. Cells to be contacted may betreated to promote assay conditions, such as selective enrichment,and/or solubilization. In these methods, target cells can be detectedusing in vitro assays that detect adenoviral proliferation, which arestandard in the art. Examples of such standard assays include, but arenot limited to, burst assays (which measure virus yield) and plaqueassays (which measure infectious particles per cell). Propagation canalso be detected by measuring specific adenoviral DNA replication, whichare also standard assays.

The following examples are provided to illustrate but not limit theinvention.

EXAMPLES Example 1 Adenovirus Vector Comprising E1A UnderTranscriptional Control of a Hypoxia Responsive Element and a PSA-TRE

General Techniques

A human embryonic kidney cell line, 293, efficiently expresses E1A andE1B genes of Ad5 and exhibits a high transfection efficiency withadenovirus DNA. To generate recombinant adenovirus, 293 cells wereco-transfected with one left end Ad5 plasmid and one right end Ad5plasmid. Homologous recombination generates adenoviruses with therequired genetic elements for replication in 293 cells which provide E1Aand E1B proteins in trans to complement defects in synthesis of theseproteins.

The plasmids to be combined were co-transfected into 293 cells usingcationic liposomes such as Lipofectin (DOTMA:DOPE.sup.TM., LifeTechnologies) by combining the two plasmids, then mixing the plasmid DNAsolution (10 .mu.g of each plasmid in 500 .mu.l of minimum essentialmedium (MEM) without serum or other additives) with a four-fold molarexcess of liposomes in 200 .mu.l of the same buffer. The DNA-lipidcomplexes were then placed on the cells and incubated at 37.degree. C,5% CO.sub.2 for 16 hours. After incubation the medium was changed to MEMwith 10% fetal bovine serum and the cells are further incubated at37.degree. C., 5% CO.sub.2, for 10 days with two changes of medium. Atthe end of this time the cells and medium were transferred to tubes,freeze-thawed three times, and the lysate was used to infect 293 cellsat the proper dilution to detect individual viruses as plaques.

Plaques obtained were plaque purified twice, and viruses werecharacterized for presence of desired sequences by PCR and occasionallyby DNA sequencing. For further experimentation, the viruses werepurified on a large scale by cesium chloride gradient centrifugation.

Adenovirus Vectors in which E1A is Under Transcriptional Control of aCell Status Specific TRE

An adenovirus vector containing a hypoxia response element (HRE) wasgenerated. CN796, an adenovirus vector in which E1A is under the controlof a composite TRE consisting of an HRE and a PSA-TRE, was made byco-transfecting CN515 with pBHG10. CN515 was constructed by inserting a67 base pair fragment from HRE eno1 (Jiang et al. (1997) Cancer Research57:5328-5335) (SEQ ID NO:1; FIG. 2) into CN65 at the BglII site. CN65 isa plasmid containing an enhancer and promoter from the human PSA gene,consisting of an enhancer from −5322 to −3738 fused to a PSA promoterfrom −541 to +12. This is the PSA-TRE contained within plasmid CN706.Rodriguez et al. (1997) Cancer Res. 57:2559-2563.

Virus Growth In Vitro

Growth selectivity of recombinant adenovirus is analyzed in plaqueassays in which a single infectious particle produces a visible plaqueby multiple rounds of infection and replication. Virus stocks arediluted to equal pfu/ml, then used to infect monolayers of cells for 1hour. The inoculum is then removed and the cells are overlayed withsemisolid agar containing medium and incubated at 37.degree. C. for 10days. Plaques in the monolayer are then counted and titers of infectiousvirus on the various cells are calculated. The data are normalized tothe titer CN702 (wild type) on 293 cells.

1. An adenovirus vector comprising an adenovirus gene undertranscriptional control of a transcriptional regulatory element (TRE)comprising a cell status-specific Tre.
 2. The adenovirus vector of claim1, wherein the adenovirus gene is essential for viral replication. 3.The adenovirus vector of claim 2, wherein the adenovirus gene is anearly gene.
 4. The adenovirus vector of claim 2, wherein the adenovirusgene is a late gene.
 5. The adenovirus vector of claim 3, wherein theadenovirus early gene is E1A.
 6. The adenovirus vector of claim 3,wherein the adenovirus early gene is E1B.
 7. The adenovirus vector ofclaim 3, wherein the adenovirus early gene is E4.
 8. The adenovirusvector of claim 1, wherein the cell status-specific TRE is human.
 9. Theadenovirus vector of claim 1, wherein the cell status-specific TREcomprises a hypoxia responsive element (HRE).
 10. The adenovirus vectorof claim 9, wherein the HRE comprises SEQ ID NO:1.
 11. The adenovirusvector of claim 1, wherein the cell status-specific TRE comprises a cellcycle specific element.
 12. The adenovirus vector of claim 11, whereinthe cell cycle-specific element is from the E2F-1 gene.
 13. Theadenovirus vector of claim 1, wherein the cell status-specific TREcomprises a heat-inducible element.
 14. The adenovirus vector of claim1, further comprising a cell type-specific TRE.
 15. The adenovirusvector of claim 14, wherein the cell type-specific TRE is prostate cellspecific.
 16. The adenovirus vector of claim 15, wherein the prostatecell-specific TRE is a PSA-TRE.
 17. The adenovirus vector of claim 1,further comprising a transgene under transcriptional control of a secondcell status-specific TRE.
 18. An adenovirus vector comprising anadenovirus gene under transcriptional control of a TRE comprising a cellstatus-specific TRE and a cell-type specific TRE.
 19. The adenovirusvector of claim 18, wherein the adenovirus gene is an early gene. 20.The adenovirus vector of claim 19, wherein the adenovirus early gene isE1A.
 21. The adenovirus vector of claim 20, wherein the cellstatus-specific TRE comprises an HRE and the cell-type specific TRE is aPSA-TRE.
 22. The adenovirus vector of claim 21, wherein the HREcomprises SEQ ID NO:1 and the PSA-TRE comprises nucleotides about 503 toabout 2086 of SEQ ID NO:3 and nucleotides about 5285 to about 5836 ofSEQ ID NO:3.
 23. A composition comprising an adenovirus vector ofclaim
 1. 24. The composition of claim 23, further comprising apharmaceutically acceptable excipient.
 25. A host cell comprising theadenovirus vector of claim
 1. 26. A method of propagating adenovirusspecific for cells which allow a cell status-specific TRE to function,said method comprising combining an adenovirus according to claim 1 withthe cells, whereby said adenovirus is propagated.
 27. A method forconferring selective cytotoxicity on a target cell, said methodcomprising contacting a cell which allows a cell status-specific TRE tofunction with an adenovirus vector of claim 1, whereby the vector entersthe cell.
 28. A method for suppressing tumor growth comprisingintroducing the adenovirus vector of claim 1 into a tumor cell whichallows a cell status-specific TRE to function, wherein introduction ofthe adenovirus vector results in suppression of tumor growth.